Human Sirt-1: Molecular Modeling and Structure-Function Relationships of an Unordered Protein

Human Sirt-1: Molecular Modeling and Structure-Function Relationships of an Unordered ProteinIda Autiero1., Susan Costantini1,2,3.*, Giovanni Colonna1,3

1 CRISCEB (Interdepartmental Research Center for Computational and Biotechnological Sciences) Second University of Naples, Naples, Italy, 2 CROM (Oncology Research

Centre of Mercogliano) ‘‘Fiorentino Lo Vuolo’’, Mercogliano, Italy, 3 Department of Biochemistry and Biophysics, Second University of Naples, Naples, Italy

Abstract

Background: Sirt-1 is a NAD+-dependent nuclear deacetylase of 747 residues that in mammals is involved in variousimportant metabolic pathways, such as glucose metabolism and insulin secretion, and often works on many differentmetabolic substrates as a multifunctional protein. Sirt-1 down-regulates p53 activity, rising lifespan, and cell survival; it alsodeacetylases peroxisome proliferator-activated receptor-gamma (PPAR-c) and its coactivator 1 alpha (PGC-1a), promotinglipid mobilization, positively regulating insulin secretion, and increasing mitochondrial dimension and number. Therefore, ithas been implicated in diseases such as diabetes and the metabolic syndrome and, also, in the mechanisms of longevityinduced by calorie restriction. Its whole structure is not yet experimentally determined and the structural features of itsallosteric site are unknown, and no information is known about the structural changes determined by the binding of itsallosteric effectors.

Methodology: In this study, we modelled the whole three-dimensional structure of Sirt-1 and that of its endogenousactivator, the nuclear protein AROS. Moreover, we modelled the Sirt-1/AROS complex in order to study the structural basisof its activation and regulation.

Conclusions: Amazingly, the structural data show that Sirt-1 is an unordered protein with a globular core and two largeunordered structural regions at both termini, which play an important role in the protein-protein interaction. Moreover, wehave found on Sirt-1 a conserved pharmacophore pocket of which we have discussed the implication.

Citation: Autiero I, Costantini S, Colonna G (2009) Human Sirt-1: Molecular Modeling and Structure-Function Relationships of an Unordered Protein. PLoSONE 4(10): e7350. doi:10.1371/journal.pone.0007350

Editor: Dafydd Jones, Cardiff University, United Kingdom

Received August 2, 2009; Accepted September 14, 2009; Published October 8, 2009

Copyright: � 2009 Autiero et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The funding is from the Institute in which this project was carried out. Funders had no role in study design, data collection and analysis, decision topublish, or preparation of the manuscript. IA is supported by Doctorate in Computational Biology, CRISCEB, Second University of Naples, Naples, Italy.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

. These authors contributed equally to this work.

Introduction

The sirtuin family is widely distributed from archaea and

eubacteria to eukaryotes and seven different homologous proteins

are found in the humans that show a central highly conserved

region, defined as the catalytic core [1]. In comparison to other

proteins of the family, Sirt-1 presents two large regions, i.e. the

amino and carboxyl terminals, that are missing in all the other

sirtuins. Sirt-1 is a NAD+-dependent deacetylase closely related to

yeast Sir2, the first gene discovered in sirtuin family, which has

NAD+ dependent class III histone deacetylase activity [2]. Sites of

phosphorylation and SUMOylation consensus were recently found

also in the amino- and in carboxyl-terminal regions, and these

were proposed having regulation and localization functions [3].

Experimental data support the Sirt-1 implication in processes

including chromatin remodelling, transcriptional silencing, chro-

mosomal stability, cell cycle progression, apoptosis, autophagy,

metabolism, growth suppression, inflammation, and stress re-

sponse [4]. In fact, the Sirt-1 regulation activity occurs through the

deacetylation reaction of various and different substrates such as

p53 [5], forkhead box class O (FOXO) transcription factors [6],

peroxisome proliferator activated receptor (PPAR)g co-activator

1a (PGC-1a) [7], nuclear factor (NF)-kB and others, which are

closely linked to some age-related diseases [5]. Also Sirt-1

stimulates eNOS activity and increases the endothelial NO. Its

inhibition in the endothelium of arteries inhibits endothelium

dependent vasodilation and decreases bioavailable NO [8].

Moreover, it was seen that Sirt-1 is a negative modulator of

adipogenesis by docking with the nuclear receptor co-repressor

(NcoR) [9] and induces a decrease of pro-inflammatory cytokine

release [10] and a promotion of carcinogenesis [11] by the

negative control of Nuclear factor-kB (NF-kB). Experimental data

demonstrated that the inhibition of NF-kB in hepatocytes in vivo

promotes hepatocarcinogenesis.

Kaeberlein et al. demonstrated firstly the anti-aging effects of

Sir2 showing that in Saccharomyces cerevisiae the integration of extra

copies of Sir2 extended lifespan up to 30% [12]. Similar effects of

Sir2 were subsequently observed in C. elegans and Drosophila

melanogaster [13–14]. In fact, the overexpression of Sir-2 [15]

increased lifespan up to 50% in C. elegans but in Drosophila, an extra

copy of the Sir2 gene extended lifespan in female and male by 29

and 18%, respectively.

PLoS ONE | www.plosone.org 1 October 2009 | Volume 4 | Issue 10 | e7350

Because Sirt-1 deacetylates non histone proteins, including

various transcription factors, it is involved in the control of

important biological mechanisms. Through its catalytic activity, it

exhibits diversified functions in cell type-specific manner, which

have pathophysiological implications in cancer, obesity, inflamma-

tion and neurodegenerative diseases [4,16–17]. Its modulation leads

an increase in mitochondrial biogenesis, and an improvement of

glucose metabolism in mitochondria but also in skeletal muscle and

adipose tissues [18]. These evidences suggest that Sirt-1 could be a

novel target to treat metabolic disorders such as type 2 diabetes.

Numerous experimental data [2,19–20] have shown the

modulation of the catalytic activity of Sirt-1 exerted through its

allosteric effectors. Kim et al (2007) demonstrated that the nuclear

protein AROS is an endogenous activator of Sirt-1 which

increases its activity interacting with the allosteric site. Accordingly

with the functional relevance of Sirt-1 activation, recent works

focused on its interaction with small allosteric effectors [19–21].

In particular, the phenolic compound, named resveratrol, is the

first found allosteric activator, that has been reported to extend

lifespan in yeast [21], Caenorhabditis elegans, Drosophila [22] and

rodents [20]. It improves the metabolism and glucose tolerance, as

well as the overall physical performance in various stress tests [7].

However recent efforts have identified new compounds signifi-

cantly more potent than resveratrol [19–20].

Even if the substrates and the effects of Sirt-1 activation have

been well characterized, no structural information about its

activation and regulation is known.

On the basis of the biological importance of this protein, we

focused our attention to create a model of the whole structure of

human Sirt-1 in order to understand its interaction with the

allosteric effectors and to propose the molecular basis of its

activation and regulation.

Results

Modelling of the catalytic core and allosteric site of Sirt-1The best model has been obtained for the catalytic core of

human Sirt-1 using as template the structures of human Sirt-2

[23], Hst2 from Saccharomyces cerevisae [21] and Sir2-Af1 from

Archaeoglobus flugidus [22] is shown in Figure 1. This model has

94,9% residues in the most favoured regions and a Prosa Z-score

of 8,7. These values, compared also with those of the template

structures, indicate that a good quality model has been created.

The catalytic core appears well structured in agreement with its

low propensity to the disorder, as shown in Figure 2. Secondary

structure predictions made by JPRED program agree with the

structure of catalytic core obtained by comparative modeling

(Figure 3). The core (from residue 244 to 498) is well structured. The

model (Figure 1) shows the two structural domains typical of the

sirtuin family of which the first one is a Rossman fold with the

characteristic b-a-b motives of NAD proteins, and the second one is

a smaller sub-domain where the zinc is located. At the interface

between domains there is the binding site for NAD. The NAD

molecule fits in a specific pocket constituted of a hydrophobic patch

Figure 1. Model of human Sirt-1. The different structural/functional regions are shown in different colors: in white the N-terminal region, in cyanthe allosteric site, in green the catalytic core and in magenta the C-terminal region. The region of the zinc binding (down on the right) shows the fourtetrahedrically coordinated cysteines.doi:10.1371/journal.pone.0007350.g001

Human Sirt-1 Structure


https://www.researchgate.net/publication/5882275_Active_Regulator_of_SIRT1_Cooperates_with_SIRT1_and_Facilitates_Suppression_of_p53_Activity?el=1_x_8&enrichId=rgreq-2ae1e5e4-c15b-4dff-a793-e54df0819050&enrichSource=Y292ZXJQYWdlOzI2ODczMzI4O0FTOjEwNDgwMjk2OTE5NDUwOUAxNDAxOTk4Mzg0NDA2

on the small sub-domain, and a hydrophilic patch on the large

domain. In particular, the NAD molecule presents the adenine and

ribose moieties inside the pocket, as described for other sirtuins

[22–25], but the nicotinamide group is near to the residues 269–295

of human Sirt-1. These residues (indicated with a white arrow in

Figure 1) represent another conserved region that forms a flexible

loop near the pocket. This loop was proposed as a ‘‘frontwall’’ [25]

of the C-site, or a ‘‘ceiling’’[25] on the pocket. Its flexible

organization allows a structural rearrangement of the catalytic

domain during the NAD binding, which seems essential for the

catalysis. This central role is demonstrated by the orientation of this

region in the crystallized sirtuins. In fact, the crystallized complexes

with the NAD+ molecule show a same order orientation, but

without NAD, this loop region shows a more poorly defined or

disordered orientation [21,23]. This suggests that the NAD+binding influences the orientation of this flexible loop by triggering

the assembly and disassembly of the C pocket [25] as well as the

loop’s orientation is a measure of the accessibility into the pocket for

the nicotinamide or other small inhibitors [1].

The zinc ion is tetrahedrally coordinated by the thiol groups of four

Cys residues (at positions 371, 374, 395, 398) which are folded into a

single structural unit. This tetrad of cysteine residues are that is broadly

conserved in the small sub-domain of all the sirtuin family [23]. and this

small zinc-binding domain (see Figure 1) is thought to play a role in

substrate-specific binding by the sirtuin proteins [26]. In the classic zinc

finger, one zinc atom is bound to two cysteines and two histidines while

in this case we have a Cys4-Zn. This structure is composed by about 30

residues included in a flexible structural environment that sterically

favours the tetrahedrally coordination of the cysteines.

The allosteric site (from residue 181 to 243) is straddling

between the N-terminal and the compact globular core of the

protein and shows an all-alpha structure composed by four alpha

helices in agreement with secondary structure and disorder

predictions (Figure 2b and 3).

Both the catalytic core and the allosteric site have compact and

globular shapes according to the definition of globularity recently

published [27] with a score of 5.1 and 4.3, respectively.

The two models were linked, as reported in Methods, by a

flexible loop and subjected to molecular dynamics simulation. The

state of equilibrium was reached after 8 ns simulation. The

structure remained very stable during the whole simulation time,

as confirmed by all the indicators commonly used to analyse MD

simulations (Fig. 4A). In particular, the Rossman fold, as well as

the smaller domain and the four helices of the allosteric site were

well conserved during the simulation. Only two flexible loops, i.e.

the final loop of the allosteric site and the ‘‘frontwall’’ in the

catalytic domain, presented some fluctuation. An evaluation of the

distance between the allosteric site and the catalytic core was

obtained in terms of center of mass distances along the trajectories.

This distance decreased of 17 A after 10 ns of simulation and was

maintained constant during the last 5 ns of simulation.

Modeling of the N-terminal and C-terminal regionsVarious programs for predicting the secondary structure of

globular proteins were unable to have a good consensus for the N-

terminal and C-terminal segments of Sirt-1. They have differently

predicted in amount and in sequence strings the scattered presence

of non alfa and non beta regions in both terminal zones. This

observation raised the suspect that the Sirt-1 could be an

unordered protein and more appropriate algorithms were used.

The N-terminal and C-terminal regions were found largely

unordered by two specific structural tests. DISOPRED, a software

devoted to the search of unordered regions predicted long

segments in N-terminal region (1–150 and 160–182) as well as

in C-terminal region (510–580, 585–640 and 680–740). Moreover,

the Anchor program, in addition, was also able to predict the

presence of definite protein binding regions [28] in the unordered

segments (Figure 2 and Table S1). The N-terminal and C-terminal

regions were modelled as reported in Methods and resulted made

of six very short helices inserted with seven short b-strands and of

five short helices inserted with five short b-strands, respectively.

The remaining large part of residues are in irregular conforma-

tions according to the predictions.

The two modelled regions were independently subjected to

molecular dynamics simulations with the same protocol used for

the allosteric and catalytic sites. The N-terminus reached a stable

equilibrated state after 8 ns and the C-terminal after 4 ns of

simulation, respectively. Both models looked very stable (Fig. 4B

and C) and the secondary structures present in both models were

well kept during the simulation time.

Complete model of Sirt-1We created a whole model of the human Sirt-1 by comparing

modelling using as templates the previously obtained models of N-

terminal, allosteric site, catalytic site and C-terminal site. The

model was minimized according to our recent papers [29].

The resulting model has 87.8% residues in the most favoured

regions and a Prosa Z-score of -6.57.

The various structural parts that compose the global architec-

ture of the protein are shown in Figure 1. The N-terminal and C-

terminal regions are positioned behind the catalytic core, but the

groove site of Sirt-1 is exposed to the solvent to receive its

substrates. Distances among the centers of mass of catalytic core,

N-terminal and C-terminal regions were evaluated. In details, the

catalytic core-N-terminal distance resulted to be 38 A, and that

between the catalytic core and C-terminal of 48 A. The catalytic

core exchanges 2 hydrogen bonds and 8 salt bridges with the N-

terminal site, and 5 hydrogen bonds and 13 salt bridges with the

C-terminal site.

These values confirm that the three regions have distinct

structural roles even if one can easily hypothesize that their very

Figure 2. Predictions of disorder regions. Top - The disorderpredictions (blue) made by DISOPRED for the N-terminal region (a), theallosteric site (b), the catalytic core (c) and the C-terminal region (d).Bottom - The prediction of disordered binding regions (black) made byAnchor.doi:10.1371/journal.pone.0007350.g002



Figure 3. Primary and secondary structures of Sirt-1 and AROS. a) catalitic core (from residue 244 to 498), b) allosteric site (from residue 181to 243) and c) AROS. Helix and beta strands regions are shown in red and cyan, respectively. Moreover, JPred predictions are reported.doi:10.1371/journal.pone.0007350.g003



close relationships suggests an involvement in the functional

activities of the Sirt-1. In fact, the allosteric site is positioned

between the N- terminus and the catalytic core in the best position

to regulate the enzymatic activity of Sirt-1. Moreover, the N-

terminal and C-terminal sites are positioned in a region that does

not prevent the catalytic core activity, and this confirms that they

have a role of regulation and localization of Sirt-1.

AROS modelAROS is the endogenous protein that is able to increase the

Sirt-1 activity [2], interacting with its allosteric site. Its model was

obtained by fold recognition strategy and presents an all-alpha fold

composed from 4 alpha helices in agreement with the secondary

structure predictions made by JPred [30] (Figure 3C) and with the

structural class prediction made by PRECLASSPRO server [31].

The AROS model was subjected to molecular dynamics

simulations by using the protocol reported in the Methods. A

stable state was reached after 7 ns of simulation time and was kept

constant up to the end of the dynamics (Figure 4D).

Sirt-1/AROS complexAROS is the endogenous protein that is able to increase the

Sirt-1 activity [2], interacting with its allosteric site. To investigate

the best binding-groove of Sirt-1 for allosteric effectors we used the

principal docking servers (i.e. PATCHDOCK, GRAMMX,

CLUSPRO) to obtain a set of possible complexes. We firstly

Figure 4. Molecular dynamics results. RMSD evolution and analysis of the secondary structures during the molecular dynamics.doi:10.1371/journal.pone.0007350.g004



selected the complexes having AROS near to the allosteric site

because experimental truncation data have suggested that some

amino acids of the allosteric site are important for the binding of

allosteric activators [19]. It is worthy of note that in the most part

of simulated complexes, AROS is placed in the region between the

N-terminal site and the allosteric site (Figure 5). For a more

detailed analysis, we selected two of the best complexes for each

set, in terms of energy binding, interface ASA, number of atoms

and residues at the interface, number of hydrogen bonds and salt

bridges (see Table 1). Moreover, we compared, in terms of

physical-chemical and geometric properties, the exposed residues

of Sirt-1 to the binding pocket in each complex to identify

pharmacophore features. As shown in Figure 6, the binding

groove presents basic residues at the top of the cavity, acidic

residues at its bottom and hydrophobic residues as edges.

The best Sirt-1/AROS complex was obtained using the

CLUSPRO web server [32] that uses one of the best docking

algorithm tested in CAPRI experiments with a success rate of about

71% [32]. The good quality of this complex is also suggested by the

very low value of its binding free energy (210.19 Kcal/mol),

calculated by the DCOMPLEX server [33]. For this complex, we

have evaluated the interacting residues, the number of interchain

H-bonds and salt bridges and the interface surface area (Tables 2

and 3). The AROS-Sirt-1 complex shows that AROS is located

behind to the catalytic groove. This can be considered one of the

most favourable structural site to increase the enzyme activity by

conformational changes without penalizing the correct interaction

between Sirt-1 and its substrates. In particular, AROS and Sirt-1

chains should form one H-bond and 6 salt bridges at their

interaction surface. At the interface Sirt-1 also exposed four

aromatic residues, one positively charged residue and five negatively

charged residues. The interface region of AROS is composed by six

and three positively and negatively charged residues, respectively.

These data suggest that the predominant interaction between Sirt-1

and AROS is on electrostatic basis and that the four aromatic

residues structurally closely in Sirt-1 might play an important role to

Figure 5. Model of human Sirt-1/AROS complex.doi:10.1371/journal.pone.0007350.g005

Table 1. The selected complex obtained using CLUSPRO, PATCHDOCK and GRAMMX server.

Complexes Binding free energy Interface ASA Interaction Residues H-bonds Salt Bridges

CL2 29.96 1023.49 34 4 24

CL10 210.19 973.9 30 1 29

PD7 210.51 1231.39 34 7 39

PD12 28.92 1230.1 39 4 29

GX2 210.73 1471.8 48 5 11

GX6 212.65 1332.27 40 4 12

For each one are reported the value of: energy binding (Kcal/mol), interface ASA (A2), number of atoms and residues at the interface, number of hydrogen bonds andsalt bridge are reported.doi:10.1371/journal.pone.0007350.t001



favour the stacking interactions with other allosteric activators, as

well as organic compounds.

Molecular dynamics of Sirt-1/AROS complexThe Sirt-1/AROS complex was subjected to molecular

dynamics simulations with the same protocol previously used.

The complex reaches a stable equilibrated state after 7 ns of

simulation and after that it remains stable. Even the secondary

structures were kept enough stable during the simulation.

We have compared the conformation of AROS/Sirt-1 complex

before and after the molecular simulation to verify how the mutual

interactions changed. We inserted an acetyl-lysine and a NAD

Figure 6. Analysing the Sirt-1/AROS binding groove. The surface of Sirt-1 binding groove of the selected complexes between AROS and Sirt-1,obtained using CLUSPRO (CL10 and CL2), PATCHDOCK (PD7 and PD12) and GRAMMX (GX6 AND GX2) servers. The colours correspond to the chemicalproperties of each residue. The acid residue are coloured in red, the basic residue are coloured in blue, the hydrophobic residues are coloured inorange and the hydrophilic residues are coloured in green. The oval lines indicate the basic top and the acid bottom, respectively.doi:10.1371/journal.pone.0007350.g006

Table 2. Analysis of the best Sirt-1/AROS complex in terms of interface surface area (A2), interchain H-bonds and number ofinteraction residues and salt bridges.

Interface ASA Interchain H-bonds Interaction residues Salt bridges

Sirt-1 973.90 1 30 6

AROS 906.00 1 25 6

doi:10.1371/journal.pone.0007350.t002

Table 3. List of interaction residues between AROS and Sirt-1.

Interaction residues

Sirt-1 MET1; ALA2; ASP3; LEU7; GLU161; ASP166; SER169; HIS170; ALA171; SER172; SER173; SER174; ASP175; TRP176; PRO184; TYR185;PHE187; VAL188; HIS191; LEU192; ILE194; GLY195; THR196; ASP197; THR219; TRP221; GLN222; ILE223; TRP624; ARG627; VAL628.

AROS ASP24; HIS25; LEU26; ARG27; GLU42; SER43; VAL44; GLN46; GLN47; ILE48; LEU49; ARG50; GLN51; ARG53; VAL62; LYS64; LYS66;LYS69; ALA70; THR73; VAL74; GLU77

The charged residues are evidenced in bold and those aromatic are also underlined.doi:10.1371/journal.pone.0007350.t003



molecule in the AROS/Sirt-1 complex using as template the

crystallographic structure of Saccharomyces cerevisae Sirt-1. Then, we

focused our attention on the catalytic groove. We noted that,

during the molecular dynamics, a conformational change of the

protein occurs mainly in active site region. In details, the residues

His 363, Asn 346, Ser 265, Gly 261 and Pro 447, indicated as

important for its catalytic role [34], are sterically changed (see

Figure S1). Also Pro 447, that makes Van der Waals contacts with

the aliphatic portion of the acetyl-lysine side chain and may affect

the positioning of residues that contact the acetyl group, is more

close to the Acetyllisine while four other residues (His 363, Asn

346, Ser 265, Gly 261) that affect the NAD orientation,

approaches to the NAD.

Therefore these results could indicate that the molecular

dynamics improves the interaction between Sirt-1 and AROS by

some structural parameters (see Table S2) even if longer time scale

will be used for studying this type of long range coupling.

Discussion

Human sirtuin-1, an oxidative stress-response and chromatin-

silencing factor, is an unordered protein. About half of its sequence

appears to be in a disordered state with long flexible poorly

structured regions observed at N- and C-terminus. The modeling

has also evidenced a large central globular part very well structured

made by two close regions, the catalytic core and the allosteric site.

Therefore, the protein can be considered composed by four

different regions: N-terminal domain, allosteric site, catalytic core

and C-terminal domain. The catalytic core is a central highly

conserved structured region common to all the sirtuin family [1]

which has the role of catalyzing the NAD+-dependent deacetylation

reaction, involved in various nuclear events such as transcription,

DNA replication, and DNA repair. The core is made by a well

organized Rossman fold typical for NAD-dependent proteins. In

details, the acetyl lysine substrate has been proposed to bind in a

narrow channel that terminates near to the nicotinamide ribose.

Moreover, the binding of acetyl-substrate is believed to mediate

bending of the nicotinamide ring of NAD+ in a strained

conformation also referred to productive NAD+ binding, promoting

the cleavage of the ribosyl–nicotinamide bond [21,24].

In this domain we find an atom of Zinc tetrahedrically

coordinated with four cysteine residues also present in the other

members of the sirtuin family. The allosteric site is a small

structural domain made of four helices and interacting with the

core. It is sited in a structural location where it can easily exert a

control of the catalytic activity by conformational changes. Several

articles report the presence of activators modulating the functional

activity of the human sirtuin. One of the activators, the resveratrol,

decreases the Michaelis constant of SIRT1 for both the acetylated

substrate and NAD+, and increases cell survival by stimulating

SIRT1-dependent deacetylation of p53 [35]. Moreover, a small

nuclear protein, AROS (Active Regulator Of Sirtuin), has been

found to be the first known endogenous active modulator of

SIRT1 which directly regulates SIRT1 function. AROS enhances

SIRT1-mediated deacetylation of p53 both in vitro and in vivo,

and it inhibits p53-mediated transcriptional activity. It is

interesting to observe that AROS was unable to inactivate p53

when was used an AROS-binding-defective SIRT1 mutant. This

clearly indicates a direct interaction of AROS with a specific site of

Sirt-1. Our best model of AROS presents an all-alpha fold

composed from 4 alpha helices. We found that AROS binds the

same allosteric site of some small synthetic compounds (not shown;

manuscript in preparation) proposed as putative therapeutics for the

treatment of type 2 diabetes [2,19].

The catalytic core and the allosteric site make a central

structured domain (about from residues 181 to 498), compact and

globular as assessed by various globularity indices.

Moreover Sirt-1 presents two long disordered structural

segments at the terminal regions, missing in the other six

homologous proteins belonging to the same family. The N-

terminal region is 180 residues long and that C-terminal 249.

These regions were predicted largely disordered by DISOPRED

program that resulted one of the best algorithm for the accuracy of

disorder prediction in CASP7 [36–37]. Figure S2 shows the

secondary structure of the best model of Sirt-1 calculated by DSSP

algorithm that evaluates the W and C dihedral angles of each

residue and the backbone Hydrogen bonds. From this table as one

can see both termini are characterized by numerous and large

unstructured regions even if in the C-terminal more structured

segments are presents.

Many proteins or protein domains show an intrinsic inability to

form a well defined tertiary structure. This property is encoded in

their sequence owing to the local depletion of typically buried

amino acid residues as well as enrichment of typically exposed

amino acid residues (about 40%) (see Table S3). Amino acid

residues located in highly mobile regions of protein also possess the

smallest volumes and molecular weights as well as the lowest

hydrophobicities and the highest flexibility (see Figures S3, S4, S5

and S6). Only in this way the protein can be so dynamically

flexible to minimize unfavorable pairwise contacts.

In the last years it has become evident that there is a large

number of proteins that do not require a stable structure even

under physiological conditions in order to fulfil their biological role

[38–40]. The importance of protein disorder is underlined by the

abundance of partially or fully disordered proteins that are

encoded in higher eukaryotic genomes [36] and are involved in

many important biological functions [38], which complement the

functional repertoire of globular proteins [41]. Disordered

segments often act as flexible linkers between folded domains in

multidomain proteins [38] and their function is often that of

binding specifically other proteins, DNA or RNA through a

process, termed coupled folding and binding, that involves a

transition disordered-ordered with stable secondary and tertiary

structural elements [38]. This ‘‘coupled binding and folding’’

confers several functional advantages in certain types of molecular

interactions that often are essential for signaling processes. The

high propensity of their residues to stay disordered makes these

regions predominantly not structured and this can favour their

putative functionality.

In fact, recent papers have shown the importance in Sirt-1 of

various phosphorilations sites, nuclear localization signals (NLS)

and nuclear export signals (NESs, amino acids ) that were mainly

found in the terminal regions [3,42,43]. These authors suggested

that these regions seem to be involved in regulating enzyme

activity and have the peculiarity of being present only in Sirt-1.

Moreover, this fact also suggests that C- and N-terminal regions of

the human Sirt-1 might be involved in a more fine regulating role

in order to exploit its biological mechanisms. In the disordered

regions specific protein binding sites were identified by using

Anchor program [28]. The prediction of these binding sites is

based on estimating the energy content in free and in the bound

states, and identifying segments that are potentially sensitive to

these changes. In particular, Anchor program has predicted in

human Sirt-1 fourtheen disordered binding regions of which four

located in the N-terminal domain, two in the allosteric site and

other eight in the C-terminal domain (Figure 2-down and Table

S1). The two disordered binding regions predicted in the allosteric

site agree with those evidenced in Sirt-1/AROS complex (see



Table 3). All these data indicate that human Sirt-1 is an unordered

protein and that its terminal domains can play different roles. The

inherent flexibility of the two termini suggests that the protein has

a malleable interface that can allow the binding of several partners

or adopt different conformations, as manifested by its high binding

capability.

Moreover the flexibility and the open structure of Sirt-1 termini

could also favour the binding of phosphorylating proteins in order

to activate regulation processes mediated by phoshorylation [3]. In

fact, very recently the presence of phosphorylation sites located in

the amino- and in carboxyl-terminal regions were found [3].

Therefore, in order to assess the ability of human Sirt-1 to be

regulated by phorphorylation [43] and if there are the structural

features inducing the interaction with phosphorylating proteins,

we have evaluated the solvent accessibility of each residue,

suggested as a putative site of phosphorylation [3]. Our data

indicate that Ser14, Ser173, Ser538, Ser 539, Ser540, Thr 544

and Ser747 are largely exposed to the solvent (Figure 7)

confirming that these residues have a high capability to be

phosphorylated. If one considers the sequence position of Ser and

Thr residues they are almost located in the N- and C- termini:

Ser14, Ser26 and Ser27 are inserted in the segment 1–33

predicted by Anchor as a protein binding site. The residues

Ser47 and Ser 159 are also included in the predicted segments 43–

49 and 133–161, respectively, while the remaining Ser169,

Ser173, Ser174, Thr196 and Thr 219 are very close to the above

predicted segments. This suggests that these residues are actually

involved in some way in the control of protein-protein binding

and, as a consequence, in the control of the Sirt-1 function. The

Sirt-1/AROS complex explains well this view by suggesting that

the interaction between the two proteins occurs primarily through

the involvement of the N-terminal segment with the achievement

of greater compactness of the complex and the presence of

changes that propagate to the active site in order to modulate the

function. Work is in progress to simulate the presence of phosphate

at various sites in the unordered regions in order to assess their

structural or functional effects during the formation of the

complex. The numerous and different structural features of

unordered proteins such as conformational heterogeneity, second-

ary structural propensities, and tertiary contacts within disordered

protein states can in principal generate many different interaction

modes in proteins. Frequently these proteins mediate in signaling

networks dynamic protein interactions that exhibit unusual

binding characteristics, such as multisite dependence and ultra-

sensitivity. Such interactions are frequently modulated by

phosphorylation, which requires disorder in the target protein

both for optimal kinase accessibility and for subsequent accessi-

bility of the binding motif [44,45]. Therefore this binding appears

to require multiple sites to be phosphorylated, suggesting a binding

mode in which multiple phosphoepitopes engage a single receptor

site in dynamic equilibrium. These observations allows to

hypothesize a binding mode in which the multiple sites found in

Sirt-1 might engage the putative receptor sites of AROS (5 Ser and

4 Thr residues) in a dynamic equilibrium. Moreover, Sirt-1

contains numerous additional phosphorylation sites remote from

the targeting regions, making their participation in the complex

unlikely. It is possible, however, that they may serve as decoy sites

that compete with the key binding sites for phosphorylation by the

targeting kinase [46].

Finally, a detailed study of the binding patches between AROS

and Sirt-1 has shown that in Sirt-1 there is a conserved

pharmacophore pocket composed by basic residues at the top of

the cavity, acidic residues at its bottom and hydrophobic residues

as edges. Moreover we have focused our attention on the presence

of five aromatic residues (i.e. Trp176, Tyr185, Phe187, Trp221,

Trp624) in the pocket that could be involved in putative stacking-

interactions. These data could explain the high affinity of the

allosteric site for synthetic activators containing aromatic rings and

hydrophobic anchor points. Investigations are in progress to study

the Sirt-1 interaction also with small allosteric effectors that have

been recently identified [19].

Methods

Modelling of catalytic and allosteric sites of human Sirt-1The three-dimensional model of catalytic site of human Sirt-1

(UniProt code: Q96EB6, region 244–498) was performed by

comparative modelling strategy using the template structures of

human Sirt-2 (PDB code: 1J8F chain A) [23], Hst2 from

Saccharomyces cerevisae (PDB code: 1Q1A chain A) [21] and

Sir2-Af1 from Archaeoglobus flugidus (PDB code: 1M2G chain A)

[22] because the percentage of sequence identity between these

proteins and human Sirt-1 was equal to 44%, 38% and 30%,

respectively. Protein sequences were aligned with CLUSTALW

[47]. The MODELLER9v5 program [48] was used to build 10

full-atom models of catalytic site, we used the ProsaII program to

check the fitness of the sequences relative to the obtained

structures and to assign a scoring function, and the PROCHECK

program [49] to evaluate their stereochemical and structural

packing quality. Secondary structures were assigned by the DSSP

program [50]. Secondary structure predictions were performed

with Jpred [29] server but structural class predictions were made

by using PRECLASSPRO server [31]. The globularity of the best

model was evaluated according to our recent work [27].

Moreover, the three-dimensional model of allosteric site of

human Sirt-1 region 181–243 was performed using the template

structure of a hexokinase from Sulfolobus Tokodaii by compar-

ative modelling (PDB code: 2E2N) [51]. As the sequence identity

between this Sirt-1 region and the homologous template model

was lower than 30% (i.e. 26%), we used a procedure strategy in

agreement with the rules recently reviewed to improve the quality

of the modelling results at low target-template sequence similarity

[52]. Allosteric and catalytic sites have been connected by using

the Builder module of Insight II and the related model was

subjected to molecular dynamics simulations.

Figure 7. Analysis of phosphorilated sites. The Solvent accessi-bility profile for the residues belonging to putative phosphorilation site,valuated using Surface program (red line) and ASAview server (blueline). The number indicates the sequence position and the one lettercode identifies the amino acid. The figure also reports the segmentalposition of residues in the protein.doi:10.1371/journal.pone.0007350.g007



Modelling of N-terminal and C-terminal regions of humanSirt-1

The BLAST research [53] has found in databases only protein

structures related to small sequences similar to the N-terminal and

C-terminal regions of human Sirt-1 [region 1–180 and 499–747,

respectively]. The prediction of unordered regions in the protein

was made by Disopred server [36]. Moreover, a prediction of

protein binding sites in the unordered regions was made by

Anchor program that identifies specific binding regions undergo-

ing disorder-to-order transition using a general disorder prediction

method IUPred based on the assumption that disordered proteins

have a specific amino acid composition that does not allow the

formation of a stable well-defined structure [28]. The prediction of

binding sites is based on estimating the energy content in free and

in the bound states, and identifying segments that are potentially

sensitive to these changes.

Therefore these regions were modelled by using a new

approach combining fold recognition and comparative modelling.

The preliminary models for N-terminal and C-terminal regions of

Sirt-1 were obtained by fold recognition strategy using the Fugue

and SAM-T06 servers [54–55]. Then, these models with the

crystallographic structures suggested by BLAST were used as

template for applying the comparative modelling strategy in order

to obtain the complete models for both regions. In details, we used

as template the following structures deposited in PDB: 1W36

(577–625), 1QHZ (1–34) for the N-terminal region and 1TYC

(76–101) for C-terminal region.

In the obtained models the loop regions were refined using the

LOOPY module of Jackal package [56]. LOOPY appeared to

yield the best results for loop modelling, with models that are on

average of 2–8% better than those generated by other programs

[52]. The models obtained for N-terminal and C-terminal regions

of Sirt-1 were subjected to molecular dynamics simulations.

Molecular dynamics simulationsMD simulations were performed with GROMACS software

package (v3.3.1) [57]. Models of different Sirt-1 regions were put

in cubic boxes filled with SPC216 water molecules and

GROMOS43a1 was selected as force-field. In order to optimize

the system, the models were previously subjected to energy

minimization and position restraints cycles. The simulations were

carried out with periodic boundary conditions by adding sodium

ions in order to the net electrostatic charge of the system is zero.

The bond lengths were constrained by the all atoms LINCS

algorithm. Particle Mesh Ewald (PME) algorithm was used for the

electrostatic interactions with a cut-off of 0.9 nm, according to

recent papers [29]. Simulations were conducted at neutral pH

where the tritable groups of His, Glu and Asp residues are

unprotonated. All simulations were run for 15 ns at room

temperature (300 K) coupling the system to an external bath.

GROMACS routines were utilized to check the trajectories and

the quality of the simulations.

Simulation of complete model of Sirt-1The whole model of human Sirt-1 was obtained by comparing

modelling using as template the models obtained for N-terminal

region, allosteric site, catalytic site, and C-terminal region and the

same procedure and programs described above. This model was

minimized by using 500 steps of energy minimization under

conjugate gradient algorithm to optimize side chain conformations

and avoid sterical clashes according to the commonly used

procedure [29,58–59].

Modelling and Simulation of Sirt-1/AROS complexThe three-dimensional model of AROS (UniProt code:

Q86WX3, region 47–123) was performed by fold recognition

strategy using the Fugue server [54]. The model obtained for AROS

was subjected for 15 ns to molecular dynamics simulations using the

protocol reported above to assess its conformational stability.

To simulate the Sirt-1/AROS complex we used the docking

web server CLUSPRO [32] that resulted the best docking

program in CAPRI experiments with a success rate of about

71% [32]. The models were selected by evaluating some features

and parameters. The ‘‘Protein–Protein Interaction Server’’ [60]

were used to identify the amino acids at the interface and to

evaluate their solvent accessibility.

Moreover, the binding free energy between the different chains

was calculated by using the DCOMPLEX program [33].

Supporting Information

Table S1 Regions predicted as protein binding sites by Anchor

program

Found at: doi:10.1371/journal.pone.0007350.s001 (0.03 MB

DOC)

Table S2 Analysis of interaction between AROS and Sirt-1

before and after MD


DOC)

Table S3 Amino acid composition of Sirt-1


DOC)

Figure S1 Details of catalytic groove before and after molecular

dynamics are shown. We reported in pink and green the carbon

atoms related to Sirt-1 before and after dynamics but N, O and H

atoms always in blue, red and white, respectively. The Acetil-

lysine, NAD and Sirt-1 residues are evidenced with labels.


DOC)

Figure S2 Secondary structure of the whole Sirt-1 structure

assigned by DSSP program. The N-terminal and C-terminal region

sequences are reported in green and blue, respectively. The helices

and beta-strands are indicated in red and cyan, respectively.


DOC)

Figure S3 Flexibility plot for Sirt-1 sequence. Ordinate reports

the value of Hydrophobicity x Volume obtained with a shifting

window of 5 according to Ragone et al. Protein Eng. 1989

2(7):497–504. Abscissa reports the residue position.


DOC)

Figure S4 Average area buried. Lower values indicates higher

exposures of residues. The graph shows that the residues in the

globular part of the protein are in average more buried than the N

and C termini. In particular, residues in the N-terminus are in

average more exposed.


DOC)

Figure S5 The average molecular weight of residues with a

shifting window of 5. The graph shows that the compact globular

core is made in average of high molecular weight residues while the

N- and C- termini are made of low molecular weight residues and

thus smaller residues are located in the more fluctuating or flexible

structural regions. It is interesting to note the highly fluctuating



values in the C-terminal region in agreement with the presence of

more structured segments in respect to the N-terminal region.


DOC)

Figure S6 Ramachandran Plot


DOC)

Author Contributions

Conceived and designed the experiments: SC GC. Performed the

experiments: IA. Analyzed the data: IA SC GC. Wrote the paper: IA

SC GC.

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Human Sirt-1: Molecular Modeling and Structure-Function Relationships of an Unordered Protein

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